PROGRAM NARRAIVE / ABSTRACT A key role of the TME in cancer progression, treatment resistance, and as a target for therapeutic intervention is increasingly appreciated. Noninvasive in vivo EPR-based spectroscopy and imaging of tissue oxygenation (pO2), extracellular pH (pHe), redox, glutathione (GSH) and interstitial inorganic phosphate (Pi) provide unique insights into biological processes in the TME, and may serve as a tool for preclinical screening of anticancer drugs and optimizing TME-targeted therapeutic strategies. In this competitive renewal application, our new directions are built on knowledge and discoveries made during the previous funding period. There are two goals of our R01 renewal proposal. First is to further advance paramagnetic probes and magnetic resonance technology with a focus on improving probes biocompatibility, functionality, and developing multifunctional imaging modalities. Second is to utilize these advances and knowledge accumulated using spectroscopic modalities in the initial R01 project period to obtain further insights into the role of TME in cancer progression, and to test our hypothesis on the roles of interstitial inorganic phosphate in tumorigenesis. The specific aims are: (SA1) To advance molecular multifunctional EPR-based imaging technology and paramagnetic probes. The biocompatible derivatives of multifunctional trityl HOPE probe sensitive to acidity (pHe), oxygen (pO2) and phosphate (Pi) in Extracellular tissue microenvironment, and dual function nitroxide pH & redox probes will be optimized for concurrent multifunctional imaging using cutting edge imaging modalities, rapid scan EPR imaging and Overhauser-enhanced magnetic resonance imaging. (SA2) To perform molecular imaging of chemical TME as the mammary tumors progress to malignancy. Multifunctional spatially-resolved TME profiling will validate morphological and gene expression-based staging in a mouse model of breast cancer and will map tumor regions with different phenotypes. Tissue samples from the areas with the chemical TME characteristics of a malignant phenotype will be isolated and the TME study will be complemented with immunohistochemical, biochemical, and genetic tissue analysis. (SA3) To study the effect of phosphate in TME on tumor progression and anticancer therapy. Under SA3, we will test the hypothesis that a low phosphate diet and pharmacological intervention with glycolysis-affecting drugs will decrease interstitial Pi and normalize other TME, thereby slowing down tumor progression and augmenting effectiveness of anticancer therapy. In summary, the developed multifunctional imaging techniques and probes will broaden the scope of preclinical EPR allowing for mapping of physiologically relevant tissue parameters in various disease models in cancers and beyond. New knowledge on stage-specific TME evolution during tumor progression is required to optimize TME-targeted anticancer therapies. It will also provide a scientific basis to evoke public awareness of high content of the phosphate- based modifiers in the processed food and the potential health risk.